The use of Dunaliella salina microalgae as bioreactors allows to produce gold nanoparticles with anticancer activity by means of green chemistry. The method has been reported for the synthesis of gold nanoparticles using extract of D. salina. This green synthesis technique involves using bright sunlight that we consider as poorly controlled condition. The mechanism of nanoparticles toxicity is not yet elucidated. The toxic effect might arise either from toxic intermediates production during bioreduction of gold ions or from physical adsorption of bioactive molecules on nanoparticles surfaces. This research aimed to optimize gold nanoparticles biosynthesis using aqueous extract of D. salina (AED) and to clarify cytotoxic properties of synthesized nanoparticles. The synthesis of nanoparticles involved heating the reaction mixture and was carried out without using sunlight exposure. The fluorometric alamar blue-based toxicity tests with mammalian cell cultures HeLa and Vero were conducted. The following pollutants were tested: biogenic nanoparticles (AED-AuNPs), 15 nm citrate-capped gold nanoparticles stabilized by aqueous extract of D. salina (Cit-AuNPs@AED), 15 nm citrate-capped gold nanoparticles stabilized by polyethylene glycol (Cit-AuNPs@PEG). We have shown that heating the reaction mixture during bioreduction of HAuCl4 by AED led to the formation of gold nanoparticles with average diameter 12.1±4.1 nm. It has been shown that AED-AuNPs exerted a selective cytotoxic effect on the cancer cell line HeLa. Kidney epithelial cell line Vero appeared to be much more resistant to AED-AuNP than HeLa cells over testconcentration range 50-400 mg Au/L. Cit-AuNPs@AED were found to be non toxic. This evidence indicate that the toxicity of biogenic nanoparticles is likely to be associated with biotransformation of D. salina metabolites during the bioreduction of chloroaurate ions.
Gold nanoparticles are intensively studied in biomedicine. Assessment of their biocompatibility is highly important. Currently there is lack of evidence, concerning nanotoxicity of ultrasmall gold nanoparticles < 5 nm. Existing data are rather contradictory. The aim of that study was to evaluate the toxicity of 2 nm colloidal gold, using microalga Dunaliella salina. Cellular barriers of that microalga are very similar to animal cells so it might be considered as a valuable model for nanotoxicity testing. Chlorophyll content as a test–function was used. Spectrophotometric method for chlorophyll determination in vivo in suspensions of D.salina cultures was applied. Calculated EC50 48h value of ionic gold was 25.8 ± 0.3 mg Au/L. EC50 value of phosphine-stabilized gold nanoclusters was 32.2 ±1.1 mg Au/L. It was not possible to calculate EC50 for 15 nm citrate gold nanoparticles, as they were non-toxic at all concentrations tested. These results are confirmed by fluorescent –microscopic monitoring of the same probes. It was shown that 10-fold growth of phosphine-stabilized gold nanoparticles (from 2.3 ± 0.9 nm to 21.1 ± 7.5 nm) led to 7-fold decrease of their toxicity.
The application of gold nanoparticles (GNPs) for laser-induced cell transfection has been studied intensively during the past decade as efficient and gentle alternative to well-established molecule delivery methods like lipid-based transfection or electroporation. The method is based on temporal increase of membrane permeability induced by laser irradiation of GNPs attached to cell membranes. Although this approach is attractive due to high throughput and easy usability, it is not free from serious drawbacks related to random adsorption of GNPs during preincubation of cells with GNPs. This stage can affect the optoporation results because of potential nanoparticle toxicity, thus leading to decreased delivery efficiency and to low reproducibility of independent optoporation runs. Herein, we suggest a novel GNP-mediated laser transfection technique based on immobilized gold nanostars (GNSs) that are adsorbed on microplate wells and act as a plasmonic surface. The HeLa cells are grown directly on the monolayer of immobilized GNSs followed by CW NIR laser irradiation. We used the propidium iodide (PI) as a model transfecting agent to monitor simultaneously the delivery of PI into HeLa cells and their viability. These proof-of-the-concept experiments demonstrated enhanced penetration of PI into irradiated cells as compared to untreated ones.
KEYWORDS: Nanoparticles, Gold, Lymphatic system, Tissues, Spleen, Bone, Plasmonics, In vivo imaging, Blood, Particles, Biomedical optics, Medical research
Currently, the usage of gold nanoparticles as photosensitizers and immunomodulators for plasmonic photothermal therapy has attracted a great attention of researches and end-users. In our work, the influence of prolonged peroral administration of gold nanoparticles (GNPs) with different sizes on the morphological changes of hematopoietic and lymphoid organs was investigated. The 24 white outbred male rats weighing 180-220 g were randomly divided into groups and administered orally for 30 days the suspension of gold nanospheres with diameters of 2, 15 and 50 nm at a dosage of 190 μg/kg of animal body weight. To prevent GNPs aggregation in a tissue and enhance biocompatibility, they were functionalized with thiolated polyethylene glycol. The withdrawal of the animals from the experiment and sampling of spleen, lymph nodes and bone marrow tissues for morphological study were performed a day after the last administration. In the spleen the boundary between the red and white pulp was not clearly differ in all experimental groups, lymphoid follicles were significantly increased in size, containing bright germinative centers represented by large blast cells. The stimulation of lymphocyte and myelocytic series of hematopoiesis was recorded at morphological study of the bone marrow. The number of immunoblasts and large lymphocytes was increased in all structural zones of lymph nodes. The more pronounced changes were found in the group with administration of 15 nm nanoparticles. Thus, the morphological changes of cellular components of hematopoietic organs have size-dependent character and indicate the activation of the migration, proliferation and differentiation of immune cells after prolonged oral administration of GNPs.
The wide application of nanotechnologies in medicine requires the careful study of various aspects of their potential safety. The effects of prolonged peroral administration of gold nanoparticles on morphological changes in lymphoid organs and indicators of peripheral blood of laboratory animals were investigated in experiment. The gold nanospheres functionalized with thiolated polyethylene glycol sizes 2, 15 and 50 nm were administered orally for 15 days to outbred white rats at a dosage of 190 μg/kg of animal body weight. The standard histological and hematological staining were used for morphological study of lymphoid organs and bone marrow smears. The size-dependent decrease of the number of neutrophils and lymphocytes was noted in the study of peripheral blood, especially pronounced after administration of gold nanoparticles with size of 50 nm. The stimulation of myelocytic germ of hematopoiesis was recorded at morphological study of the bone marrow. The signs of strengthening of the processes of differentiation and maturation of cellular elements were found in lymph nodes, which were showed as the increasing number of immunoblasts and large lymphocytes. The quantitative changes of cellular component morphology of lymphoid organs due to activation of migration, proliferation and differentiation of immune cells indicate the presence of immunostimulation effect of gold nanoparticles.
We synthesized and characterized gold nanostars and their silica-coated derivatives with 7- to 50-nm shell thicknesses as contrast agents for optical imaging. The scattering and absorption coefficients of the nanoparticles (NPs) were estimated by means of collimated transmittance and diffuse reflectance/transmittance analyses. The contrasting properties of the nanostructures were studied in optical coherence tomography glass capillary imaging. The silica-coated nanostars with the thickest shell have higher scattering ability in comparison with bare nanostars. Viability assays confirmed weak in vitro toxicity of nanostructures at up to ∼200-μg/mL concentrations. We showed real-time visualization of nanostars in both agarose and cultured cells by analyzing the backscattering signal using a conventional laser confocal microscope. The signal intensity detected from the silica-coated NPs was almost 1.5 times higher in comparison with bare nanostars. To the best of our knowledge, this is the first time that conventional laser confocal microscopy was applied in combined scattering and transmitted light modes to detect the backscattered signal of gold nanostars, which is useful for direct monitoring of the uptake, translocation, and accumulation of NPs in living cells.
Plasmon-resonant nanoparticles attached to cell membranes, under laser treatment can temporarily increase membrane permeability. In this paper, the influence of continuous-wave and pulsed (nanosecond) laser irradiation on living cells incubated with gold nanoparticles was investigated. Gold nanospheres, nanostars, and nanorods with different functionalization were used as plasmonic agents. The dependence between increase of medium temperature on the irradiation time was showed for nanostars and nanorods with different surface properties. Cells samples incubated with gold nanorods showed the highest temperature increase. Feasibility of cell optoporation by the use of gold nanospheres with variable functionalization was demonstrated. The cell membrane permeability was successfully enhanced as shown by the uptake of the fluorescent dye upon nanosecond laser treatment. Toxicity of the nanoparticles was estimated by MTT assay.
In this paper, plasmon-resonant nanostructures, such as gold nanostars and their silica-coated composites, were used for enhancement of OCT image contrast of water flows in glass capillaries. The contrasting properties of the synthesized nanostars and nanocomposites with silica shell thickness of about 5 nm and 50 nm were compared in the framework of capillary stasis model. The most intensive signal was detected from the nanocomposites with the thickest silica shell. The nanocomposites were characterized by optical spectroscopy and electron microscopy. Nontoxicity of nanostars and nanocomposites up to ~ 3 mg/mL concentration was showed by MTT assay suggesting practical applications of the nanostructures for bioimaging.
The oral administration of gold nanoparticles at dose 190 mg /kg animal body weight during 8 days leads to changes in
the cellular composition of the mesenteric lymph nodes, the severity and reversibility of the reaction was determined by
the size of gold nanoparticles. The cellular composition of the lymph nodes after the introduction of gold nanoparticle
size of 1-3 nm for 8 days corresponded the control values, indicating the absence of immunotoxicity. Duration of
recovery of cellular composition of the lymph nodes after administration of gold nanoparticles with size 15 nm and 50
nm does not exceed 21 days. It can be concluded that 15 nm and 50 nm nanoparticles have no immunotoxic action at oral
administration.
In this work, two types of nanocomposites, silica-coated nano-sea-urchins and silica-coated gold nanostars, were
fabricated. CTAB-coated nano-sea-urchins with an average size of about 100 nm demonstrate an absorption peak near
600-700 nm and stability in aqueous suspension. CTAB was exchanged with m-PEG-SH by an intermediate PEG layer.
A layer of silica was synthesized on the nano-sea-urchins surface with thickness of about 20 nm. Nanostars with an
average size of about 60 nm with a number of thin sharp branches were fabricated and functionalized by PVP to improve
their stability. PVP-coated nanostars were used in optical coherence tomography experiments to show their contrasting
properties. After silica-coating, stable and monodispersed nanoparticles with silica shell thickness about 60 nm were
obtained. Nontoxicity of the silica-coated nanostars at least until the concentration of nanoparticles about 400 μg/mL was
showed by fluorescent cell viability assay using propidium iodide. Extinction coefficient of the gold nanostars and
nanocomposites was estimated by a spectrophotometer system in collimated transmission regime.
Manufacturing processes for different types of hermetically coated fibers are described. Optical and mechanical
properties of metal and carbon coated fibers are compared. Prospects of application of both types of hermetically coated
fibers in special applications are discussed.
Light-induced inactivation of dynamic response of somatic frog nerve on electrical pulsed excitation was study ex vivo. The light-sensitive Indocianin Green has been used on photodynamic induced inactivation of the processes generation nerve pulses. Inactivation of consequence action potential of somatic frog nerve using excitation of electrical pulsed was achieved by irradiation with diode laser light in a IR spectral region (λ=810 nm, P~1W/cm2) in the case of Indocianin green. It was discovered that Indocianine green decrease of the amplitude compound action potential of the ensemble neurons. Experiments show effective destruction of cancer cells of ear, mouth and skin by local injection of plasmon resonant gold nanoshells and semiconductor laser (810 nm) irradiation. For destruction such tumors pulse duration was not less than 1microsecond and pulse separation 10 at average power density 1-3 W/sm2 and energy density 100-200 J/sm2
We describe applications of colloidal gold nanoparticles and silica/gold nanoshells to photothermal therapy and X-ray diagnostics of cancer exemplified by spontaneous tumor of cats and dog. It is shown that the contrast of X-ray images of a cat mammalian tumor can be increased significantly by injection of 3-5-nm gold nanoparticles. We also present preliminary results of a complex approach to treatment a dog mouth cavity melanoma by using immunotherapy together with laser photothermolysis enhanced with silica/gold nanoshells.
The study is aimed at evaluation of diagnostic potential of plasmon-resonant nanoparticles as contrast agents for various techniques based on backward diffuse light scattering from biotissues. We consider the theoretical spectra of diffuse backscattering from a plane layer of gold nanospheres embedded into a plane layer of scattering dielectric spheres that mimic a strongly scattering sample of a biotissue. It is shown that the backscattering spectra contain information about 35-nm gold spheres provided the location depth is less than 600 mkm. The simulated color characteristics resemble the measured characteristics of a rat skin. The measured diffuse reflection spectra of 35-nm gold colloid are close to the spectra of 70/15-nm silica/gold nanoshells although the plasmon resonance wavelengths (520 and 630 nm) differ significantly.
We report on the first application of silica-gold nanoshells to a solid-phase dot immunoassay. The assay principle is based
on staining of a drop (1μL ) analyte on a nitrocellulose membrane strip by using silica/gold nanoshells conjugated with
biospecific probing molecules. Experimental example is human IgG (hIgG, target molecules) and protein A (probing
molecules). For usual 15-nm colloidal gold conjugates, the minimal detectable amount of hIgG is about 4ng. By contrast,
for nanoshell conjugates (silica core diameter of 70 nm and gold outer diameter of 100 nm) we have found significant
increase in detection sensitivity and the minimal detectable amount of hIgG is about 0.5 ng.
A new variant of a technique for in vivo production of antibodies to various antigens with colloidal-gold nanoparticles as carrier is discussed. With this technique we obtained highly specific and relatively high-titre antibodies to different antigens. The antibodies were tested by an immunodot assay with gold nanoparticle markers. Our results provide the first demonstration that immunization of animals with colloidal gold complexed with either haptens or complete antigens gives rise to highly specific antibodies even without the use of complete Freund's adjuvant. These findings may attest to the adjuvanticity of gold nanoparticles itself. We provide also experimental results and discussion aimed at elucidation of possible mechanisms of the discovered colloidal-gold-adjuvanticity effect.
We report on the time-dependent light scattering from single gold nanospheres (diameter about 90 nm) and nanorods (15x25 nm). Unlike the recent report by S&diaero;nnichsen and Alivisatos (Nano Lett. 5, 301-304, 2005), we observed the dynamic modulation of scattering intensity by using unpolarized dark field microscopy equipped with a digital camera. The changes in light scattering intensity from gold nanorods were caused by 3-dimensional rotational diffusion by contrast to 2-dimensional polarized-scattering modulation observed previously. In the case of 90-nm gold nanospheres, the light scattering modulation was related to the translational diffusion only. By using T-matrix method and Rayleigh approximation, we describe the time-dependent variations in light scattering intensity caused by rotation of nanorods at several scattering and orientation geometry. Finally, we present preliminary data on real-time visualization of active interaction between the living rat peritoneal macrophages and gold nanorods.
We describe synthesis process of an oligonucleotide-functionalized colloidal gold marker CG-l5-T28, its optical properties and interaction with poly(A) in solution and on a solid-phase substrate. The marker is a complex of 15 nm diameter colloidal gold nanoparticles with covalently attached 5'-thiolated 28-base oligothymidine macromolecules. A positive hybridization reaction of the marker with poly(A) is observed by solid-phase analysis on hanging a spot color (from red to blue ) or on appearance of a red dye in dot-blot test as compared to control experiments with poly(U) target. The principles of spectrophotometric monitoring all stages of the marker preparation and application of spectrophotometry to detection of the polynucleotide hybridization in vitro are described. Experimental data were compared with theoretical calculations based on Mie theory for 2-layer model of gold core in polymeric shell with imaginary part of refractive index that typical for the real absorption spectra of NA. To explain the aggregation of CG-15-T28 caused by interaction with poly(A) in solution, we suggest a new model differing from a standard model of cross-linker binding.
We report on a synthesis procedure and optical properties of composite Au-Ag spherical and rod-like nanoparticles. The synthesis protocol is based on a seed-mediated growth in the presence of soft templates in micellar aqueous solution of ionic surfactant (CTAB). Variation of Au/Ag molar ratio allows one to produce nanorods (NRs) with different aspect ratio. The disadvantage of the method is formation of appreciable amount of spherical nanoparticles. To separate rod-like particles from spheres and surfactant, we used a fractionation procedure that involves centrifugatiori of samples in the density gradient of glycerol. The separated NRs were suspended in water or 25% glycerol solutions and their extinction and differential light scattering (at 900) spectra were recorded for 450-850 ni-n wavelengths. Theoretical spectra were calculated by T-matrix method as applied to randomly oriented gold cylinders with semispherical ends. The simulated spectra for water and glycerol suspensions can be brought in close agreement with experimental observations if the aspect ratio is used as a fitting parameter. We discuss also the absorption and light scattering contribution to the total extinction spectra and deviation of the exact solution from the classical electrostatic approximation by Gans.
Three procedures for determining the average size of colloidal gold (CG) nanoparticles are described. The first procedure is the dynamic light scattering (DLS) method. The others are based on the estimation of extinction or differential static light scattering (DSLS, at 90 degrees) peak positions. Experimental studies were carried out with four samples of CG (the average DLS-diameters were equal to 15, 20, 25, and 30 nm). In the case of DSLS, the angular slope of the calibration curve "peak position versus particle size" was 2.4 times greater than the extinction curve slope. In experiments with controlled polydispersity, two different colloid mixtures were used. Extinction spectra of samples were almost identical, whereas the DSLS spectra showed noticeable differences with a red shifted maximum. The theoretical simulations by the Mie theory are in agreement with these experimental observations. The DSLS technique seems more reliable and accurate in CG particle sizing, as compared with the usual absorption spectroscopy.
In this work, we report on the optical properties of colloidal gold bioconjugates obtained by attaching protein molecules or oligonucleotide probes to the surface of the particles. Addition of complementary components to the bioconjugate probe results in a specific assembling of conjugates and can be monitored by light scattering and extinction spectra. To this end, we have developed a special attachment to the Specord M-40 spectrophotometer and a corresponding measurement procedure called by us differential light scattering spectroscopy. This method is suitable for the nearly simultaneous recording of extinction and static light scattering spectra of a simple that possesses overlapping spectral bands of absorption and light scattering.
In this paper, we report on the optical properties of aggregates formed by biospecific interactions like antigen/antibody, with one or both reaction components immobilized on gold particles. In the case of biospecific aggregation, the temporal changes in the absorption spectra differed from those recorded during rapid and slow salt aggregations. As in the case of rapid salt aggregation, the absorption peak decreased and shifted to the red part of the spectrum with simultaneous broadening. However, we did not observe the second red peak of the optical density. According to the transmission electron microscopy data, the slow, rapid, and biospecific aggregations resulted in small clusters with compact structures, branching aggregates of fractal type, and aggregates without direct conductive contacts of primary particles, respectively. It is supposed that the recorded differences in absorption spectra can be explained by the corresponding differences in aggregate structures. We have found a direct correlation between the amount of the second added protein initiating aggregation on the one hand, and the rate of spectral changes on the other. Using these spectral changes, we have plotted a calibration curve for a sufficiently rapid and technically simple quantitative test like sol-particle immunoassay.
We report on the experimental spectra of light scattered at 90 degrees by colloidal gold particles (diameter 30 nm) and aggregates built from these particles during the salt aggregation process. The time dependence of spectra in the range 350-850 nm was measured by using a luminescence attachment to Specord M-40 spectrophotometer. The aggregation of sol was accompanied by essential increasing the single particle spectrum maximum (near 580 nm), its displacement to the red part in the spectrum, and by a broadening of the long wavelength wing of extinction. To explain these findings, we used a computer diffusion-limited cluster-cluster aggregation model. The optical properties of aggregates including light scattering spectra were computed by the coupled dipole method (CDM or DDA). The bulk optical constants of metals were modified by the size-limiting effect of nanoparticles. Our calculations showed a direct correlation between cluster particle number (Nequals10-50) and intensity of scattered light. However, this correlation failed for 100-particle clusters. It can be supposed that DDA model is not adequate for accurate prediction of light scattering properties of large aggregates.
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